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OBJECTIVES Early warning scores detecting clinical deterioration in pediatric inpatients have wide-ranging performance and use a limited number of clinical features. This study developed a machine learning model leveraging multiple static and dynamic clinical features from the electronic health record to predict the composite outcome of unplanned transfer to the ICU within 24 hours and inpatient mortality within 48 hours in hospitalized children. METHODS Using a retrospective development cohort of 17 630 encounters across 10 388 patients, 2 machine learning models (light gradient boosting machine [LGBM] and random forest) were trained on 542 features and compared with our institutional Pediatric Early Warning Score (I-PEWS). RESULTS The LGBM model significantly outperformed I-PEWS based on receiver operating characteristic curve (AUROC) for the composite outcome of ICU transfer or mortality for both internal validation and temporal validation cohorts (AUROC 0.785 95% confidence interval [0.780–0.791] vs 0.708 [0.701–0.715] for temporal validation) as well as lead-time before deterioration events (median 11 hours vs 3 hours; P = .004). However, LGBM performance as evaluated by precision recall curve was lesser in the temporal validation cohort with associated decreased positive predictive value (6% vs 29%) and increased number needed to evaluate (17 vs 3) compared with I-PEWS. CONCLUSIONS Our electronic health record based machine learning model demonstrated improved AUROC and lead-time in predicting clinical deterioration in pediatric inpatients 24 to 48 hours in advance compared with I-PEWS. Further work is needed to optimize model positive predictive value to allow for integration into clinical practice.

Abstract Background People with cancer experience high rates of venous thromboembolism (VTE). Risk of subsequent cancer is also increased in people experiencing their first VTE. The causal mechanisms underlying this association are not completely understood, and it is unknown whether VTE is itself a risk factor for cancer. Methods We used data from large genome-wide association study meta-analyses to perform bidirectional Mendelian randomization analyses to estimate causal associations between genetic liability to VTE and risk of 18 different cancers. Results We found no conclusive evidence that genetic liability to VTE was causally associated with an increased incidence of cancer, or vice versa. We observed an association between liability to VTE and pancreatic cancer risk [odds ratio for pancreatic cancer: 1.23 (95% confidence interval: 1.08–1.40) per log-odds increase in VTE risk, P = 0.002]. However, sensitivity analyses revealed this association was predominantly driven by a variant proxying non-O blood group, with inadequate evidence to suggest a causal relationship. Conclusions These findings do not support the hypothesis that genetic liability to VTE is a cause of cancer. Existing observational epidemiological associations between VTE and cancer are therefore more likely to be driven by pathophysiological changes which occur in the setting of active cancer and anti-cancer treatments. Further work is required to explore and synthesize evidence for these mechanisms.

Introduction: Patients with TCE RRMM have a high disease burden and often report worsening HRQoL with additional lines of therapy. The single-arm, phase 2 KarMMa study demonstrated that ide-cel, a B-cell maturation antigen (BCMA)-directed chimeric antigen receptor T cell therapy, improved HRQoL from baseline in patients with TCE RRMM who had received ≥ 3 prior regimens (Delforge M, et al. Blood Adv 2022;6:1309-1318). Furthermore, in the phase 3 KarMMa-3 clinical trial (NCT03651128), ide-cel significantly improved progression-free survival and response compared with standard regimens in patients with TCE RRMM who had received 2-4 prior regimens (Rodriguez-Otero P, et al. N Engl J Med 2023;388:1002-1014). Here we assessed the comparative effects of ide-cel versus standard regimens on HRQoL in patients with TCE RRMM who had received 3-4 prior regimens including an immunomodulatory agent, a proteasome inhibitor, and daratumumab (an anti-CD38 monoclonal antibody) using data from the KarMMa-3 study. Methods: Patient-reported outcomes (PROs) were assessed by the European Organization for Research and Treatment of Cancer (EORTC) Quality-of-Life Questionnaire-Core 30 (QLQ-C30), EORTC Quality of Life Questionnaire Multiple Myeloma Module (QLQ-MY20), and EQ-5D-5L. The pre-specified primary domains of interest included EORTC QLQ-C30 global health status/QoL, physical functioning, cognitive functioning, fatigue, and pain; EORTC QLQ-MY20 disease symptoms and side effects of treatment; and the EQ-5D-5L visual analog scale (EQ-5D VAS). PROs were collected at screening (baseline), the day of ide-cel infusion (ide-cel) or first treatment dose (standard regimens), monthly from 2 to 28 months, and thereafter every 3 months. Comparisons were performed on least squares (LS) mean changes from baseline through 28 months between arms using constrained longitudinal data analysis (cLDA). Effect size was estimated by Hedges' g. The comparative effects of ide-cel versus standard regimens on time to confirmed improvement and time to confirmed deterioration (defined by prespecified change thresholds) were assessed via stratified Cox proportional hazards regression analyses. All P values are nominal and not adjusted for multiple testing. Statistical significance was set at P < 0.05. Results: Baseline characteristics were well balanced among the patients in the ide-cel arm (n = 167) and the standard regimen arm (n = 93), with a median age of 63 years and a median time of 4.7 years from RRMM diagnosis to study screening. Completion rates of HRQoL assessments were high over time for most visits (> 75%). At baseline, mean HRQoL domain scores were similar between arms. Overall LS mean changes from baseline to 28 months showed significant differences in favor of ide-cel versus standard regimens (nominal P < 0.05) for 11 domains (effect sizes of 0.26-0.82), and 7 (4 primary: global health status/QoL, cognitive functioning, pain, EQ-5D VAS; 3 secondary: dyspnea, insomnia, constipation) of them exceeded the prespecified MID thresholds (Table). Time to confirmed improvement was significantly shorter (nominal P < 0.05) in the ide-cel arm than in the standard regimen arm for 10 domains, including all primary domains except pain. Time to confirmed deterioration was longer in the ide-cel arm than the standard regimen arm for most domains, with significant differences in emotional functioning, cognitive functioning, and dyspnea (EORTC QLQ-C30). Finally, side effects of treatment favored the ide-cel versus standard regimen arm (EORTC QLQ-MY20) (Table). Conclusions: These patient-reported findings demonstrate that compared with standard regimens, treatment with ide-cel improved MM-relevant disease symptoms, functioning, and overall health status/HRQoL of patients with TCE RRMM in the KarMMa-3 trial. These data further substantiate the superior treatment outcomes of ide-cel over standard regimens in this patient population. Overall, treatment with ide-cel may help alleviate detriments to HRQoL in these patients with complex disease.

INTRODUCTION Richter transformation (RT) is the histologic transformation of CLL/SLL into an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). We previously reported an incidence of 0.5% per year of RT in patients with newly diagnosed CLL and an incidence of 1% per year following initiation of therapy (Parikh, BJH, 2013). It is unknown whether these incidences have changed in the novel agent era. METHODS We identified patients with previously untreated CLL/SLL in the Mayo Clinic CLL Database who were seen within 12 months of diagnosis. Only patients with biopsy-proven DLBCL with histopathology confirmation at Mayo Clinic were included. Cumulative incidence methodology was used to display the time to development of RT with death as a competing risk (both from time of initial CLL/SLL diagnosis, and from start of CLL directed therapy). We defined the time period prior to February 2014 (FDA approval of ibrutinib for CLL) as the pre-novel agent era and the time period starting February 2014 as the novel agent era. Using Cox regression analysis, we compared the incidence of RT, both after initial CLL diagnosis and after initiation of CLL therapy, between patients diagnosed with CLL in the pre-novel agent era versus the novel agent era. RESULTS Between 1/2000 and 4/2023, 3347 patients were seen at Mayo Clinic. The median age at diagnosis was 65 years (range 21-97 years), and 66% patients were male. Baseline characteristics of patients at the time of CLL and RT diagnoses between the two treatment eras were similar, including distance in miles of primary residence from Mayo Clinic ( Table 1). The median follow-up for the entire cohort was 5.6 years (9.5 years for the pre-novel agent era cohort and 3.2 years for the novel agent era cohort). A total of 82 patients were diagnosed with RT (69 in the pre-novel agent era and 13 in the novel agent era). Overall, the 1-, 5- and 10-year cumulative incidence rates for RT from the time of CLL/SLL diagnosis were 0.6% (95% CI: 0.4-0.9%), 1.8% (95% CI: 1.3-2.3%), and 3.0% (95% CI: 2.4-3.8%), respectively. Among the patients who received CLL/SLL therapy (n=1382), the 1-, 5-, and 10-year incidence rates for RT from the time of treatment start were 0.3% (95% CI: 0.1-0.8%), 3.3% (95% CI: 2.4-4.5%), and 4.9% (95% CI: 3.7-6.5%), respectively. Among 1981 patients diagnosed with CLL/SLL in the pre-novel agent era, the 1- and 5-year incidence rates for RT from the time of diagnosis were 0.6% (95% CI: 0.4-1.1%) and 2.1% (95% CI: 1.5-2.8%), respectively. Among 1366 patients diagnosed with CLL/SLL in the novel agent era, the 1- and 5-year incidence rates following CLL/SLL diagnosis were 0.5% (95% CI: 0.2-1.1%) and 1.1% (95% CI: 0.6-2.2%), respectively. The RT incidence from time of CLL/SLL diagnosis was not significantly different between the pre-novel agent and novel agent era patients (hazard ratio [HR] 1.6, 95%CI 0.9-3.0, p=0.13). Patients who received CLL/SLL treatment in the pre-novel agent era (n=926) and novel agent era (n=456) had subsequent 1-year incidence rates of RT of 0.3% (95% CI: 0.1-1.0%) and 0.2% (95% CI: 0.0-1.8%), respectively. The 5-year incidence rates for these groups were 3.7% (95% CI: 2.6-5.2%) and 1.9% (95% CI: 0.7-5.0%), respectively. Although an increased risk of RT after CLL/SLL directed therapy was observed in the pre-novel agent era compared to the novel agent era (HR 2.4, 95%CI: 0.9-6.1, p=0.07; Figure 1), this did not cross the threshold of statistial significance. Among patients diagnosed in the pre-novel agent era who developed RT (n=69), CLL treatment prior to RT included: none, n=19 (28%); chemoimmunotherapy only, n=36 (52%); and novel agent, n=14 (20%; includes 11 treated with prior chemoimmunotherapy also). In contrast, among patients diagnosed in the novel agent era who developed RT (n=13), CLL treatment prior to RT included: none, n=8 (62%); and novel agent, n=5 (38%). CONCLUSIONS Patients who received CLL/SLL directed treatment appear to have a lower risk of RT in the novel agent era. This may be driven by the fact that none of the patients who developed RT in the novel agent era received chemoimmunotherapy. Whether novel agent use for CLL/SLL suppresses the development of RT or avoids the risk associated with cytotoxic chemotherapy-induced clonal evolution requires further study. Although limited by length of follow-up, our findings challenge the notion that RT incidence will increase in parallel with CLL patients living longer with novel agent treatments.

Background: An interim analysis (data cutoff date April 18, 2022) of the international, open-label, phase 3 KarMMa-3 trial (NCT03651128) showed the chimeric antigen receptor T cell therapy idecabtagene vicleucel (ide-cel) significantly improved progression-free survival and treatment response rates, as well as HRQoL, as compared with standard regimens, in patients with triple-class exposed (TCE) RRMM who had received 2-4 prior regimens (Rodriguez-Otero P, et al. N Engl J Med 2023;388:1002-1014; Delforge M, et al., HemaSphere, 2023;7(S3):P905). The aim of the present study was to analyze the effect of ide-cel versus standard regimens (daratumumab [DARA], pomalidomide, and dexamethasone [DEX]; DARA, bortezomib, and DEX; ixazomib, lenalidomide, and DEX; carfilzomib and DEX; or elotuzumab, pomalidomide, and DEX) on HRQoL in KarMMa-3 trial participants with an extended follow-up of patient-reported outcomes (PRO) data collection (data cutoff date April 28, 2023). Methods: In KarMMa-3, HRQoL was assessed using the European Organisation for Research and Treatment of Cancer (EORTC) Quality-of-Life Questionnaire-Core 30 (QLQ-C30), EORTC Quality of Life Questionnaire Multiple Myeloma Module (QLQ-MY20), and EQ-5D-5L. HRQoL assessments were completed at screening (baseline); the day of ide-cel infusion or the first dose of standard treatment; and monthly from 2 to 28 months; and every 3 months thereafter. The prespecified primary outcomes of interest were EORTC QLQ-C30 global health status/QoL, physical functioning, cognitive functioning, fatigue, and pain; EORTC QLQ-MY20 disease symptoms and side effects of treatment; and the EQ-5D visual analog scale (EQ-VAS). Least squares (LS) mean changes from baseline through month 25 for ide-cel and standard regimens were compared by constrained longitudinal data analysis (cLDA). Effect sizes were estimated by Hedges' g. The effect of ide-cel versus standard regimens on time to confirmed improvement and deterioration in HRQoL (defined by prespecified change thresholds and sustained duration) were compared by stratified Cox proportional hazards regression. There was no adjustment for multiple testing, and all P values reported are nominal. Results: Overall, 254 patients were randomized to the ide-cel arm and 132 to the standard regimens arm. Baseline characteristics were well balanced across treatment arms; both arms had a median age of 63 years, a median of 3 prior anti-myeloma regimens, and median time from MM diagnosis to screening was 4 years. The baseline HRQoL scores were generally comparable between treatment arms. Overall LS mean changes from baseline to month 25 showed significant differences (nominal P value < 0.05) in favor of ide-cel for 18 out of 21 HRQoL domains, with effect sizes (Hedges' g) of 0.27 to 0.82 (Table). For 13 of these domains (including 6 primary domains of interest), the difference in overall LS mean change from baseline exceeded the prespecified minimum important difference (MID) threshold. Time to confirmed improvement was significantly shorter in the ide-cel arm than the standard regimens arm for 19 domains, including all primary domains of interest (Table). Time to confirmed deterioration showed trends of being longer in the ide-cel arm than the standard regimens arm for most domains and was significantly longer for emotional functioning, cognitive functioning, social functioning, dyspnea, and constipation as measured by the EORTC QLQ-C30. The time to confirmed deterioration in side effects of treatment (based on the EORTC QLQ-MY20) was significantly longer for patients in the ide-cel arm and time to confirmed improvement was significantly faster, as compared with the standard regimens arm (Table). Conclusions: In the KarMMa-3 trial, patients with TCE RRMM who had received 2-4 prior regimens, ide-cel significantly and meaningfully improved MM-relevant symptoms, functioning, and overall health status/HRQoL compared with standard regimens. Confirmed HRQoL improvements in the ide-cel arm occurred sooner than standard regimens. PRO improvements were sustained over more than 2 years, which is notable after a single infusion treatment with ide-cel. These patient-reported findings further support the benefit of ide-cel in this patient population.

INTRODUCTION We aim to identify factors that impact outcomes of venetoclax for patients with CLL treated in routine practice at a tertiary center. We report on the use of venetoclax in the most frequently encountered disease scenarios: first-line, relapsed/Bruton tyrosine kinase inhibitor (BTKi)-naïve, and relapsed/BTKi-exposed. METHODS We identified patients who received venetoclax therapy for CLL (between 4/2012-4/2023) from the Mayo Clinic CLL Database. Undetectable measurable residual disease (uMRD) was defined as <1 CLL cell per 10,000 leukocytes using 8-color flow cytometry on peripheral blood (PB) or bone marrow (BM). Overall survival (OS) was defined as the time from venetoclax start until date of death or last known to be alive. Treatment-free survival (TFS) after venetoclax was defined as the time from venetoclax start until the earliest of date of next treatment, or death. Kaplan-Meier was used to display OS and TFS. Multivariable Cox proportional hazards regression models were used to estimate associations of factors with time-to-event outcomes. RESULTS A total of 155 patients received venetoclax: firstline therapy (in combination with obinutuzumab, n=55) and relapsed CLL (n=100; 17 had relapsed/BTKi-naïve CLL, and 83 had previously received BTKi [55 with progression after BTKi], relapsed/BTKi-exposed). The median follow-up for the cohorts of first-line therapy, relapsed/BTKi-naïve, and relapsed/BTKi-exposed was 12.9 months, 37.0 months, and 27.6 months, respectively. Baseline characteristics at the time of venetoclax initiation for all patients are shown in Table 1. The median TFS for the overall cohort was 39.0 months. The median OS was 54.6 months. Among patients treated with venetoclax as first-line therapy (n=55), the 2-year TFS ( Figure 1) and 2-year OS rates were both 91%. MRD testing was performed in 28 patients and was uMRD in 23 (82%) patients (only PB assessed, n=7; only BM assessed, n=2; PB and BM assessed, n=14). Detectable MRD was identified in 3 patients, and 2 patients had discordant results (PB uMRD and BM detectable disease). Among patients treated with venetoclax in the relapsed/BTKi-naïve setting (n=17), the 2-year TFS rate was 73% ( Figure 1) and the 2-year OS rate was 100%. MRD testing was performed in 7 patients and was uMRD in all 7 (100%) (only PB assessed, n=3; only BM assessed, n=2; PB and BM assessed, n=2). The median time to first uMRD result was 14.1 months. Among relapsed/BTKi-exposed venetoclax-treated patients (n=83), the median TFS was 26.9 months ( Figure 1), and the median OS was 39.4 months.In this subgroup, the median TFS for patients with (n=55) and without (n=28) prior disease progression on prior BTKi were 22.3 and 42.3 months, respectively. Median TFS with venetoclax monotherapy (n=30) was 24.0 months, venetoclax in combination with rituximab (n=37) was 26.9 months, and venetoclax in combination with obinutuzumab (n=16) was 39.0 months. BTKi-exposed patients that were chemotherapy-naïve (n=27) and chemotherapy-exposed (n=56) had median TFS of 29.1 and 24.0 months, respectively. MRD testing was performed in 28 patients and was uMRD in 16 (57%) patients (only PB assessed, n=9; only BM assessed, n=2; PB and BM assessed, n=5). Detectable MRD was identified in 11 patients, and 1 had discordant results (PB uMRD and BM detectable disease). The median time to first uMRD result was 11.5 months. TP53 disruption, unmutated IGHV genes, older age, complex karyotype (CK; defined as more than 3 chromosomal aberrations on CpG stimulated karyotype), and disease progression on prior BTKi were associated with shorter TFS in the overall cohort on univariate analysis. TP53 disruption, older age, CK, and disease progression on prior BTKi were associated with shorter OS in the overall cohort on univariate analysis. Multivariable analysis was performed by including only those patients where all variables significant in univariable analysis were available (OS model n=65, TFS model n=53). In these models, only CK was significantly associated with shorter TFS (HR 8.5; 95%CI 2.5-29.1; P<0.001) and shorter OS (HR 4.1; 95%CI 1.2-14; P=0.03). CONCLUSIONS Patients with BTKi-exposed CLL, particularly those with prior disease progression on BTKi, had worse outcomes. Our study identified CK as one of the most important baseline predictors of adverse TFS and OS in the overall cohort of patients, supporting karyotype assessment for prognostication prior to venetoclax treatment.

Introduction: Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a rare form of large granular lymphocytic leukemia. The clinical characteristics and outcomes of patients (pts) with CLPD-NK in the literature are limited. We aim to describe the patient characteristics, laboratory features, and clinical outcomes of CLPD-NK by reporting on, to our knowledge, the largest single-center cohort of CLPD-NK in the United States. Methods: We retrospectively identified Mayo Clinic pts with CLPD-NK, defined per the WHO 5 th edition and 2022 International Consensus Classification. We conducted chart review to collect clinical and laboratory data. Overall survival (OS) was plotted using Kaplan-Meier. Time to first treatment (TTFT) was displayed using cumulative incidence methodology accounting for competing risk of death. Univariable Cox regression models investigated associations between patient characteristics and outcomes (OS and TTFT). Results: We identified 45 pts who were diagnosed with CLPD-NK between 1997 and 2022. The median age at diagnosis was 69 years (range: 25-90) and 32 pts (71%) were male. At the time of diagnosis, 10 pts (22%) had a concomitant autoimmune disorder and 9 (20%) had laboratory evidence of a serologic abnormality (monoclonal gammopathy, rheumatoid factor, etc.). In addition, 10 pts (22%) had a prior diagnosis of a hematologic malignancy and 12 (27%) had a prior diagnosis of a solid tumor. Clinically, 4 pts (9%) had B symptoms, 15 (33%) had splenomegaly, and 13 (29%) were transfusion dependent. At diagnosis, median hemoglobin (hgb) was 10.2 g/dL, with 25 pts (56%) showing a hgb <11g/dL and 8 (18%) demonstrating a hgb <8 g/dL. The median platelet count was 202x10^9/L, with 12 pts (27%) demonstrating a platelet count <150x10^9/L. The median white blood cell count was 5.8x10^9/L. The median absolute neutrophil count (ANC) was 1.2x10^9/L, with 29 pts (64%) demonstrating an ANC <1.5x10^9/L. The median absolute lymphocyte count (ALC) was 3.8x10^9/L, with 20 pts (44%) demonstrating an ALC >4x10^9/L. The median absolute abnormal NK cell count per flow cytometry was 2.2x10^9/L. Flow cytometry revealed killer cell immunoglobulin-like receptor (KIR) restriction in all pts, with 27 (61%) lacking KIR expression and 17 (39%) demonstrating KIR restriction in CD158a, CD158b, and/or CD158e. CD7 expression was present in 42 pts (93%; 31% dim/partial), CD8 expression was present in 22 (49%; 42% d/p), CD56 expression was present in 17 (39%; 23% d/p), CD57 expression was present in 26 (59%; 46% d/p), CD94 expression was present in 33 (77%; 7% d/p), and NKG2A expression was present in 21 (50%; 5% d/p). Bone marrow examination (n=30) revealed that the median number of NK infiltrate was 11% (range: 5-40%) of cellularity, with 20 demonstrating an intrasinusoidal infiltrative pattern. The median follow-up was 41 months (mo), and 23 pts required at least one line of treatment. Treatment indications included anemia (39%), neutropenia (4%), bi/pancytopenia (39%), lymphocytosis (4%), or other (13%). The median TTFT was 32 mo (95% CI 2-93 mo). Hgb <11g/dL was associated with a shorter TTFT (9.7 vs 187 mo); however, neutropenia and thrombocytopenia were not associated with TTFT. For first-line treatment, in addition to frequent steroid use, 16 pts (70%) received a methotrexate-based regimen, 6 (26%) received a cyclophosphamide-based regimen, and 1 (4%) received a cyclosporine-based regimen. The median TTFT was not reached. Of those who required systemic treatment, 9 pts required a second-line therapy. The median OS from diagnosis was 162 mo (95% CI: 84-not estimable, NE), with 33 pts alive at last follow-up (Figure 1a). Increasing age (HR 1.2, 95% CI 1.1-1.3, p=0.002) and lack of CD94 expression (HR 5.6, 95% 1.3-25.0, p=0.02) were associated with worse OS. In the 23 pts who required treatment, the median OS from treatment initiation was 84 mo (95% CI: 52-NE) (Figure 1b). Conclusions: In this large cohort of 45 pts, the majority had neutropenia and anemia, and approximately half required systemic treatment. Estimated median OS from diagnosis exceeded a decade for the entire cohort and was seven years after treatment initiation. Anemia was associated with a shorter TTFT, while increasing age and lack of CD94 expression were associated with worse OS. This rare disease warrants additional investigations to understand optimal management and improve outcomes, especially for those requiring treatment.

Background: Identification of minimal residual disease (MRD) in acute myeloid leukemia (AML) is essential for assessing risk of relapse and guiding therapeutic management. While molecular assays have streamlined AML MRD detection, flow cytometric immunophenotyping (FCI) remains the only available test modality for approximately 50% of patients lacking traceable molecular MRD targets. Unfortunately, due to the high level of expertise required, long manual analysis time and complexity of AML MRD FCI data, only a few reference laboratories offer this as an orderable test. Methods: To facilitate the analysis of AML MRD by FCI, we devised an unsupervised machine learning (ML) pipeline that intakes raw FCI data, eliminates acquisition errors (FlowCut), performs state-of-the-art clustering (PARC) and dimensionality reduction (UMAP), estimates how aberrant or different-from-normal each cluster is compared to a cohort of controls (15 donor and 15 reactive marrow aspirates), downsamples clusters with high event counts without significant loss of low-event clusters, and corrects for downsampling using an upsampling factor. This CCADDAS (Clinically-intended Clustering of All events, Dimensionality reduction, Downsampling and Aberrancy Scaling) pipeline was containerized, deployed in a cloud environment (Google Cloud Vertex AI), and used to process raw FCI files from 50 AML MRD FCI assays (3-tube/10-color panel) with 0.1% to <5% leukemic subsets, and 20 assays with no detectable leukemic subsets by prior conventional analysis. Machine learning-enhanced data files were analyzed using a commercially available FCI analysis software (Kaluza v 2.1, Beckman Coulter). Results: Cluster-informed downsampling reduced the total number of events needed to be analyzed from 1 million cells to 179,531 cells per tube on average (82% data reduction), while preserving low event subsets (clusters smaller than 5000 cells) and allowing for accurate quantitative estimates for any gated population [# events x mean (upsampling factor)]. MRD was detected in all positive cases, and absent on all negative cases when analyzed using the enhanced downsampled data (100% correlation with original data), with accurate quantitative estimates (Spearman r = 0.85). Analysis time was dramatically reduced from 73 minutes to 8 minutes per case on average (89% reduction of manual analysis time). Dimensionality reduction parameters simplified gating of lineage- and maturation-defined subsets. Cluster comparison against control samples facilitated the rapid identification of MRD subsets, based on a calculated aberrancy scale parameter that helped distinguish MRD blasts (mean ± 2SD: 0.85 ± 0.3) from benign blasts (mean ± 2SD: 0.26 ± 0.3) (p<0.0001). Conclusion: We introduce CCADDAS, an unsupervised containerized cloud ML pipeline that enhances raw AML-MRD FCI data with ML annotations, cluster-informed downsampling and comparison to normal controls to create markedly smaller and software-agnostic FCI files. CCADDAS simplifies and accelerates detection of AML-MRD in clinical diagnostics, reducing number of cells analyzed by 82% and manual analysis time by 89%, without impacting test performance. Moreover, CCADDAS can be deployed on any laboratory-developed AML-MRD assay, and its small-sized export is compatible with any clinical FCI software, computing platform and analysis strategy. Adoption of CCADDAS is likely to facilitate the implementation of AML MRD FCI analysis by more clinical laboratories.

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.

INTRODUCTION Chronic lymphocytic leukemia (CLL) and SLL represent a single entity in current classifications per the WHO-HAEM5 and ICC. Both are characterized by malignant B cells with an identical immunophenotype, but they are distinguished by the predominant site of disease burden, such as peripheral blood for CLL and nodal compartments for SLL. Most clinical trials and retrospective studies involving CLL/SLL do not separate out SLL patients. In this study, we report reasons for treatment initiation and outcomes in a cohort of SLL patients seen at our institution. METHODS Using the Mayo Clinic CLL Database, we retrospectively identified patients with SLL who received first line therapy between 12/1995 and 2/2023. Patients were classified as having SLL if they met at least one of the following criteria for diagnosis: 1) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute clonal B cell count of < 5 x 109/L; or 2) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute lymphocyte count of < 11 x 109/L (if absolute clonal B cell count was not available). Although the presence of anemia (hemoglobin < 11 g/dL) and thrombocytopenia (platelets < 100 x 109/L) are considered as exclusion criteria for a diagnosis of SLL, patients were included if these cytopenia were due to autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP) with < 50% bone marrow involvement by SLL (bone marrow biopsy confirmed). Patients treated for SLL with extranodal involvement were also included. Indications for SLL treatment were abstracted by chart review and included one of the following: 1) symptomatic or progressive lymphadenopathy and/or splenomegaly, 2) autoimmune cytopenias, 3) extranodal/direct organ infiltration; and 4) other (e.g., cryoglobulinemia, paraneoplastic, non-infiltrative renal disease such as minimal change disease). Overall survival (OS) was defined as the time from treatment start until date of death or last known to be alive. Treatment-free survival (TFS) was defined as the time from treatment start until the earliest of date of next treatment, or death. Kaplan-Meier method was used to display OS and TFS. Cox proportional hazards regression models were used to estimate associations of factors with time-to-event outcomes. RESULTS We identified 182 patients with SLL who received first line treatment. The median age at treatment start was 66 years, and 132 (73%) were male. Unmutated IGHV was identified in 60/82 (73%) patients and peripheral blood fluorescent in situ hybridization (FISH) assessed pre-treatment start showed del13q in 10%, trisomy 12 in 43%, del11q in 7%, del17p in 8%, and no abnormalities in 33%. TP53 disruption (del17p and/or TP53 mutation) was present in 7/89 (8%) patients prior to first therapy. The median follow-up after diagnosis was 6.9 years. The indications for treatment were grouped as follows: symptomatic or progressive lymphadenopathy/splenomegaly (n=130), extranodal/direct organ infiltration (n=30), AIHA/ITP (n=10), and other (n=12). For the entire cohort, the median TFS was 2.9 years and the median OS was 12.3 years. The median TFS by the treatment indication groups were 3.3, 5.1, 1.6, and 5.3 years, respectively (Figure 1). The median OS by treatment indication groups were 11.1, 10.5, 8.8, and 19.9 years, respectively. Among the 160 patients with symptomatic or progressive lymphadenopathy/splenomegaly or extranodal disease, the treatments administered include: mAb alone (n=35), chemotherapy alone (n=33), chemoimmunotherapy (n=57), targeted agent (n=33; Bruton tyrosine kinase inhibitor [BTKi] based n=28; venetoclax-based n=3, BTKi + venetoclax n=2), and other (n=2). The median TFS by treatment groups were 2.4, 2.0, 3.8, and 6.0 years (Figure 2), respectively (excluding the small “other” subgroup of 2 patients). The median OS for these groups were 9.5, 9.7, 15.0, and not evaluable, respectively. Only older age at treatment start predicted for shorter TFS (HR 1.02; 95%CI 1.002-1.038; p=0.03) and shorter OS (HR 1.05; 95%CI 1.03-1.07; p<0.01). CONCLUSIONS In this study of patients with SLL who received therapy, the most frequent indications for treatment included symptomatic lymphadenopathy and/or splenomegaly, followed by extranodal disease. Trisomy 12 was the most frequently detected abnormality on peripheral blood FISH testing. Treatment with novel agents achieved durable TFS and OS.